In Indonesia, the over-exploitation and habitat degradation of its biodiverse freshwater fish populations, has led to an urgent need for conservation of endangered fish species. A review is conducted on sustainable captive breeding practices for native Indonesian freshwater fish, with emphasis placed on the importance of ex situ conservation strategies. The key components of captive breeding such as ecological and biological research in the field; capturing, handling, and transportation of fish; selection of genetically diverse breeding stock; care and quarantine measures; feed acclimatization; and creation of reproductive facilities have been discussed. Natural, artificial, and semi-natural breeding methods have also been reviewed, and their advantages and limitations have been highlighted. The challenges in maintaining genetic diversity, managing health, ensuring successful acclimatization, and facilitating reproduction have been identified, and strategies to overcome them have been proposed. By integrating conservation and economic objectives, this review underscores the dual role of captive breeding in preserving endangered species and enhancing the ornamental fish trade, thereby contributing to the sustainable management of Indonesia's freshwater fish resources. This review adds to the literature by offering a comprehensive synthesis of sustainable captive breeding practices for native Indonesian freshwater fish, filling a critical gap in global conservation efforts and providing practical recommendations for similar initiatives worldwide.

Traditionally, sperm and embryos were studied using microscopy to assess morphology and motility. However, OMICS technologies, especially transcriptomic analysis, are now being used to screen the molecular dynamics of fertility markers at cellular and molecular levels, with high sensitivity. Transcriptomics is the study of the transcriptome - RNA transcripts produced by the genome - using high-throughput methods to understand how the RNAs are expressed. In this review, we have discussed gene contributions to sperm structure and function and their role in fertilization and early embryo development. Further, we identified miRNAs shared by sperm, oocytes, and early embryos and their roles in fertilization and early embryo development.

Pyometra is a common life-threatening inflammatory disease with a complex etiopathogenesis that develops during the diestrus stage and can be observed in elderly intact bitches. The present study evaluated five aquaporin (AQP1, AQP2, AQP3, AQP5, and AQP9) transcript abundances and immunolocalization in the uterine tissue, and investigated their relationship with uterine tissue and blood lipopolysaccharide (LPS) concentration, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activity, and nitric oxide (NO) production in dogs suffering from pyometra. The study sampled 36 client-owned intact bitches from different breeds, of which 24 cases were diagnosed with pyometra. Twelve of these bitches in the diestrus stage that presented for elective ovariohysterectomy were used as the control group. Blood samples were collected into tubes without anticoagulant for serum progesterone, LPS concentration, and antioxidant activities at the time of diagnosis. Bacteriological and tissue samples from the uteri were collected after the ovariohysterectomy. The tissue samples were used to determine antioxidant activity, and hormone and toxin concentrations. Transcript abundance of uterine AQPs were determined by qPCR, and their presence and localization were determined by by immunohistochemistry. For all pyometra samples, the bacteria isolated from the uterine swabs were Escherichia coli. Compared to the control group, AQP1, AQP2, and AQP5 were downregulated more than 2-fold, whereas AQP9 was upregulated nearly 3-fold and AQP3 was upregulated more than 4-fold in the pyometra affected uteri (P<0.05). Uterine AQP1 was moderately negatively correlated with serum LPS concentration (r=-0.568, P<0.01) and tissue NO production (r=-0.407, P<0.05). AQP5 was positively correlated with serum SOD activity (r=0.485, P<0.05) and negatively correlated with serum LPS concentration (r=-0.512, P<0.05). AQP9 was negatively correlated with tissue SOD and serum GPx activity. This is the first study to identify AQP9 transcript abundance and immunolocalization in canine uterine tissue. Uterine AQP1, AQP2, AQP3, AQP5, and AQP9 transcript abundances were altered in spontaneously developed canine pyometra while AQP transcript abundance was negatively related to serum toxin concentration, NO production, and antioxidant enzyme activity. Further studies should be conducted to determine the role of altered abundances of AQPs transcripts in pyometra pathogenesis.

Reproductive success in mammals hinges on the ability of sperm to generate sufficient energy through cellular metabolism to perform the energy-intensive processes required for fertilisation, including motility, maturation, and oocyte interactions. It is now widely accepted that sperm exhibit metabolic flexibility, utilising a combination of glycolysis and oxidative phosphorylation (supported by the Krebs cycle and other complementary pathways) to meet their energy demands. However, the preferred pathway for energy production varies significantly among species, making it challenging to map species-specific metabolic strategies, particularly in species with high metabolic flexibility, like the ram. Additionally, differences in methodologies used to measure metabolism have led to biased interpretations of species' metabolic strategies, complicating the development of liquid storage methods aimed at preserving spermatozoa by manipulating energy generation based on species-specific requirements. This review examines sperm energy requirements, current methods for assessing metabolic capacity, and the current research on species-specific metabolism. Future research should focus on establishing a standardised approach for determining metabolic preferences to accurately map species-specific strategies, a critical step before developing effective liquid preservation methods. By identifying species-specific regulatory points, strategies can be designed to temporarily inhibit metabolic pathways, conserving resources and reducing the accumulation of metabolic by-products. Alternatively, supplementation with depleted metabolites can be guided by understanding areas of excessive consumption during prolonged metabolism. Applying this knowledge to develop tailored preservation techniques will help minimise sperm damage and improve survival during in vitro processing and liquid storage, ultimately enhancing the success of artificial breeding programs.

Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current flow in response to an applied voltage. Though, these methods are limited by low throughput, as they assess mitochondrial activity at a single time point under a specific treatment condition. This study explores, for the first time, the application of the Agilent Seahorse XFp Technology to evaluate metabolism in stallion spermatozoa. This method enables real-time measurement of cellular metabolism across multiple samples or experimental conditions simultaneously. Ejaculates from eight different stallions were collected, and pools were prepared from three of them. Sperm viability and mitochondrial activity were evaluated by fluorescence microscopy, sperm motility by a computer-assisted sperm analysis system, and sperm metabolism was analysed via the Seahorse XFp analyser. Results confirmed a preference for OXPHOS over glycolysis in ATP production in stallion sperm, with mitochondria contributing significantly to total ATP generation. The Seahorse XFp Technology proved effective in evaluating equine sperm bioenergetics, offering insights into metabolic pathways critical for sperm function. In conclusion, this technology grants a new method for high-throughput analysis of sperm metabolism and quality, which could be applied to future reproductive studies in male equine fertility.

Reproductive efficiency is crucial for animal agriculture. This economically important aspect can be influenced by environmental burdens, nutritional imbalance, and gonadal or gametic malformations of genetic origin. Successful implementation of genomic-driven selective breeding in cattle depends on the reproductive performance of artificial insemination (AI) sires with valuable genomic production traits. Reproduction is traditionally viewed as a complex set of polygenic traits that are negatively impacted by using a small number of often closely related sires selected for AI due to their superior genetics. Despite recent progress, it remains difficult to define relationships between sire genome and variation in sperm phenotypes, even though several types of heritable, non-compensable sperm defects have been identified. In this review, we discuss the concept of sperm quality biomarker discovery and genomics of male fertility. We also outline a multidisciplinary genome-to-phenome approach for investigating heritable mutations and their impacts on bull fertility, sperm phenotypes and paternal contributions to early pregnancy. High-precision phenotyping requires novel, state-of-the-art instrumentation for sperm quality evaluation and development of new biomarkers of sperm quality in farm animals, with potential for incorporation into andrology-specific machine learning protocols and translation to human andrology. We conclude that reproduction is a complex phenotype that can be deciphered and explored for more precise male fertility evaluation and higher reproductive efficiency.

The objective of this study was to investigate the early follicular apoptosis in canine ovarian follicles by examining the expression of anti-apoptotic BCL-2 and pro-apoptotic BAX proteins throughout the estrous cycle associated with oocyte maturation. Follicular cells from preantral and antral follicles of varying sizes were isolated and grouped based on follicle type and estrous phase. Antral follicles underwent flow cytometry analysis, whereas preantral follicles were subjected to Western blotting. The meiotic capacity of oocytes from these different follicle types was evaluated through in vitro maturation. Statistical analysis included ANOVA and Duncan's test. Results showed fluctuations in BCL-2 and BAX levels across different follicular stages and estrous phases. BCL-2 levels increased (P<0.05) with follicular development in antral follicles, particularly during estrus, while BAX exhibited variations peaking (P<0.05) during estrus. The BCL-2/BAX ratio in antral follicles was higher (P<0.05) in estrus and diestrus compared to anestrus and proestrus. Additionally, BCL-2 and BAX proteins were detected in preantral follicles, with varying expression levels (P<0.05) across estrous phases. The BCL-2/BAX ratio in preantral follicles was highest (P<0.05) during anestrus and estrus and decreased (P<0.05) in proestrus and diestrus. Oocytes from preantral follicles did not reach the MII stage, regardless of the levels of BCL-2/BAX. Conversely, oocytes obtained from large follicles during estrus showed the highest (P<0.05) maturation percentages, which were associated with the highest BCL-2/BAX ratio. These findings provide insights into the dynamic patterns of BCL-2 /BAX in canine follicles across the estrous cycle, shedding light on their potential roles in oocyte development.

Litter size is a key trait in livestock breeding. The BMP15 and KISS1 genes have been studied in goats, but results on their association with litter size are inconsistent. The objective of this study was to employ a meta-analysis approach to investigate the genetic relationship between the BMP15 (g.735 G>A) and KISS1 (g.2540 C>T and g.2510 G>A) genes and litter size in goats. A total of five studies (including 12 breeds) were included for the g.735 G>A mutation, three studies (including nine breeds) for g.2540 C>T, and two studies (including six breeds) for g.2510 G>A in this meta-analysis. The meta-analysis was conducted under four different genetic models: recessive (GG + AG vs. AA), dominant (GG vs. AG + AA), additive (GG vs. AA) and codominant (GG + AA vs. AG) models of inheritance. Data were analyzed under either random or fixed effects models based on the estimates of I estimates. A sensitivity analysis was performed by removing one study at a time to determine the stability of the overall results. Funnel plots and the Egger regression tests were also used to assess the publication bias among studies. Significant associations (P< 0.05) were observed between the g.2540 C>T and g.2510 G>A loci and litter size in goats under the additive (SMD = -0.469, 95 % CI = -0.908 to -0.030, P-value = 0.036) and codominant (SMD = 0.147, 95 % CI = 0.003-0.291, P = 0.046) genetic models, respectively. Our results did not identify any significant association between g.735 G>A of BMP15 and litter size under the investigated genetic models.

Various prenatal factors including the number of littermates (fraternity size) and exposure to male littermate (fraternity sex ratio) during fetal period have been reported to influence postnatal fertility in the mammals. The present research was conducted to study the association of fraternity size and sex ratio with reproductive performance of nulliparous ewes and does. To this end, data associated with number of littermates, exposure to male littermate, birth weight, age at first pregnancy, as well as litter size, sex ratio of offspring, litter weight, and birth weight of female and male offspring after the first parturition retrieved from the database of sheep (n = 536 Romane and 289 Blanche du Massif Central ewes) and goat (n = 174 Alpine and 267 Saanen does) flocks. Fraternity size was negatively associated with birth weight of ewes and does (P < 0.05). Exposure to male littermate during fetal period was associated with younger age at first pregnancy and larger litter size in the does (P < 0.05), but not in the ewes (P > 0.05). Exposure to male littermate during fetal period was positively associated with the odds of male-biased litters in the ewes and does (P < 0.05). Fraternity size was positively associated with litter weight in the does (P < 0.05), but not in the ewes (P > 0.05). In conclusion, the present study showed that the number and sex of littermates during fetal period could impact postnatal reproduction of ewes and does. In this context, some associations, particularly those related to exposure to male littermate during fetal period, were only observed in does, which implicates that the effect of androgens on developmental programming of reproduction may be species-specific.

This review describes the first steps necessary to apply any reproductive biotechnology in South American camelids (SAC) semen or sperm: sample collection, evaluation and handling. In camelids, the length and position adopted for mating and the site of semen deposition have conditioned semen collection methods. The advantages and disadvantages of available collection methods are summarized. The two main drawbacks for applying assisted reproductive techniques in SAC: sperm concentration and rheological characteristics are discussed. Techniques currently available to reliably evaluate diverse sperm characteristics are described, as are different methods to improve semen handling. Finally, advances made regarding the role of seminal plasma in SAC spermatozoon physiology are addressed. Part II of the review will cover the subsequent steps of dilution and cryopreservation of samples. Current results obtained using artificial insemination (AI) in SAC will also be covered in Part II.

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

This experiment assessed pregnancy losses from day 31 of gestation to calving in lactating Holstein cows reared in tropical conditions, and evaluated if serum concentrations of haptoglobin and pregnancy-associated glycoproteins (PAGs) during early gestation differs according to pregnancy losses. Cows (708 primiparous and 844 multiparous) were assigned to an ovulation synchronization + fixed-time artificial insemination (FTAI) protocol (day -11-0 of the experiment). Pregnancy status was verified using transrectal ultrasonography on days 31, 62, 120, and according to calf birth. Blood samples were collected from all cows on day 24, and from cows diagnosed as pregnant on day 31. Pregnancy losses were greater (P < 0.01) from day 31-62 (12.8 %) and day 120 to calving (12.1 %) compared with day 62-120 (6.42 %). Pregnancy losses were greater in multiparous compared with primiparous cows from day 31-62 (17.1 vs. 9.5 %) and from 120 to calving (15.4 vs. 7.7 %). Serum PAGs concentrations on day 31 were less (P ≤ 0.03) in cows that lost the pregnancy from day 31-62 (3.57 ng/mL) and from day 62-120 (4.40 ng/mL) compared to cows that maintained the pregnancy (5.57 and 5.66 ng/mL, respectively). Cows that experienced pregnancy loss from day 31-62 had greater (P = 0.05) serum haptoglobin concentrations on day 24 (0.414 mg/mL) compared with cows that maintained the pregnancy (0.271 mg/mL). Collectively, this experiment provides novel information about pregnancy losses after day 31 of gestation in lactating Holstein cows reared in tropical environments.

Artificial insemination is an important biotechnology employed in livestock production. Production of semen products requires analysis of sperm concentration, motility and morphology. Although adequate analytical procedures are essential to ensure product quality, several multicenter studies have reported large variations in semen analysis results within and across laboratories. Differences in equipment and methodology, inconsistent training and performance testing, and lack of quality assurance programs are likely responsible for these observations. The somewhat pervasive perception that semen analysis is a trivial task must be challenged as it jeopardizes efficient semen production and germplasm utilization. This manuscript reviews the Quality Assurance (QA) procedures recommended to control results obtained during semen analysis. Reference, standard technical specifications for basic semen analysis are also described.

Mugil cephalus is a species of considerable interest for aquaculture. As a species that is not sexually dimorphic, when building a brood stock with a balanced sex ratio there is difficulty for identification of sex until the animals are quite mature. When mono-sex populations of this species is desired, as in the case of production of females for "bottarga", considerable resource expenditure could be saved if early sorting of sexes were possible to enable selection of a single sex. A recently described sex-associated loci within the follicle stimulating hormone receptor gene (fshr) was identified using genomic DNA sequencing and shown to contain some non-synonymous mutations wherein the females have a tendency to be homozygous and the males heterozygous. The loci identification method is time-consuming and somewhat expensive. We propose that the method described for the identification of the genetic sex of Mugil cephalus, prior to sexual maturation should be rapid and not require DNA sequencing. In this work, we demonstrate the utilization of one of these fshr mutations within this genetic marker in a PCR-RFLP assay. Using this new method, the loci in question shows 77.8 % partitioning with males and 88.9 % partition with females, as referenced to phenotypic sex characterized by histology, thus confirming the partitioning of the genetic marker as seen previously using DNA sequencing. Future applications of this relatively rapid and inexpensive method could contribute to the production of mono-sex farmed stock and open new opportunities for more efficient broodstock management practices in the species.

The objective of the present study was to determine the ovarian ultrasonographic findings and metabolic factors that influence the effect of human chorionic gonadotropin (hCG) treatment on the fifth day after artificial insemination (AI) in dairy cows. Thirty-seven lactating Holstein cows were assigned to two groups: the hCG group (n = 25), which received 3000 IU of hCG intramuscularly on Day 5 after AI (day of AI = Day 0), and the control group (n = 12), which received no treatment. Ovarian ultrasonography measured luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (= LBF/LTA), and dominant follicle area on Day 5. Blood tests measured plasma insulin-like growth factor-I, insulin, and metabolite concentrations on Day 5 and plasma progesterone concentrations on Days 5 and 7. LBF was greater in pregnant cows than in non-pregnant cows, and plasma Glu concentration was lesser in pregnant cows than in non-pregnant cows, but in both cases there was no interaction between group and pregnancy outcome. For plasma insulin concentration, there was an interaction between group and pregnancy outcome, with pregnant cows in the hCG group having lesser concentrations than the other groups. Logistic regression analysis showed that group and the interaction between group and plasma insulin concentration were associated with pregnancy outcome. These results suggest that the effect of hCG treatment on Day 5 after AI is related to plasma insulin concentration and is more effective in cows with lesser plasma insulin concentrations.

In this study, it was aimed to evaluate the effect of seminal plasma activity levels of enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), non-enzymatic antioxidant alpha-tocopherol (Vit-E), and also total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) on post-thaw sperm quality. As well as it was aimed to investigate the possibility of the use of OSI as a marker for the estimation of bull semen freezability. For this study, 72 ejaculates were collected from 6 bulls and separated into two aliquots. The first one was centrifuged to separate seminal plasma. The latter one was cryopreserved and stored in liquid nitrogen until analysis. Post-thaw semen quality was examined in two groups (good-freezable semen (GFS) and poor-freezable semen (PFS)) through cluster analyses based on post-thaw total motility and plasma membrane and acrosome integrity. As a result of the analyses, seminal TAS, CAT, and Vit-E values were higher (P<0.05) in the GFS group, while TOS and OSI values were higher (P<0.01) in the PFS group. We also performed an ROC curve analysis to determine whether the seminal OSI value could be used to predict semen freezeability. The area under curve (AUC) value was found as 0,70 (P=0.006). In conclusion, it has been revealed that the seminal plasma antioxidant content is responsible for the freezability of semen, and the OSI value, which can be determined by performing TAS and TOS analyses instead of looking for separate antioxidant enzymes, can be used as a marker for the estimation of post-thaw semen quality at artificial insemination centers.

Fish egg quality is very crucial in aquaculture sector for production of healthy seed. Egg yolk is an energy reservoir for growth and development of embryo. This study evaluated the biochemical composition and quality of Cirrhinus mrigala eggs at three different hatchery sites of Pakistan, (Site 1= Fish Seed Hatchery, District Pakpattan; Site 2= Sidhuwan Hatchery, Head Balloki, District Kasur; Site 3= Chenab Fish Hatchery, Rangpur, District Muzaffargarh) during induced breeding. For this, a total of 36 (18 males and 18 females) fish brooders, 12 (06 males and 06 females) from each site were utilized. Fatty acids analysis revealed significant differences among three different sites. Saturated fatty acids i.e., palmitic acid (C16:0) and stearic acids (C18:0) were higher at site 2 compared to the others sites. Monounsaturated fatty acids i.e., Oleic acid (C18:1) and polyunsaturated fatty acids i.e., Docosahexaenoic acid (DHA) (C22:6) exhibited considerably greater values at site 2 than those of other two sites. Egg mineral contents unveiled remarkable differences, particularly at site 2 indicating significantly higher mineral contents except copper (Cu) in comparison to the other sites. Significant variation exists in fertilization and hatching rates during induced spawning, with the highest values recorded at site 2. It is concluded that biochemical composition of egg especially fatty acid profile and mineral content greatly influences the embryonic development and hatching success of farm reared Cirrhinus mrigala.

Since its introduction in animal andrology, flow cytometry (FC) has dramatically evolved. Nowadays, many compartments and functions of the spermatozoa can be analyzed in thousands of spermatozoa, including, but not limited to DNA, acrosome, membrane integrity, membrane symmetry, permeability, and polarity; mitochondrial mass and mitochondrial membrane potential, identification of reactive oxygen species, ion dynamics, and cellular signaling among many others. Improved machines, many more probes, and new software are greatly expanding the amount of information that can be obtained from each flow cytometry analysis. Modern flow cytometers permit the simultaneous investigation of many different sperm compartments and functions and their interactions, allowing the identification of sperm phenotypes, helping to disclose different sperm populations within the ejaculate. Complex flow cytometry panels require a careful design of the experiment, including selecting probes (fully understanding the characteristics and properties of them) and adequate controls (technical and biological). Ideally, compensation and management of data ("cleaning", transformations, the establishment of gates) are better performed post-acquisition using specific software. Data can be expressed as a percentage of positive cells (typically viability assays), intensity of fluorescence (arbitrary fluorescence units, i.e. changes in intracellular Ca) or dim and bright populations (typically assays of membrane permeability or antigen expression). Furthermore, artificial intelligence/self-learning algorithms are improving visualization and management of data generated by modern flow cytometers. In this paper, recent developments in flow cytometry for animal andrology will be briefly reviewed; moreover, a small flow cytometry experiment will be used to illustrate how these techniques can improve data analysis.

Heat stress is caused by exposure of animals to high temperatures and humidity, outside their thermal comfort zone. This can have negative outcomes, including adversely affecting general well-being and reducing productive and reproductive performance. In males, heat stress can disrupt testicular thermoregulation, with deleterious effects on spermatogenesis and consequently, decreases in sperm quality and fertility potential. Thus, high environmental temperature is considered one of the most important factors that predisposes bulls to subfertility and has already been the subject of many studies, particularly in tropical or subtropical countries. It is essential to study effects of testicular heat stress in bulls, know the chronology of clinical and sperm findings, and understand the underlying pathophysiology. In addition, elucidating molecular mechanisms involved in heat stress and testicular function could provide the basis for effective, evidence-based strategies for selecting more thermotolerant animals. Excessive heat affects expression of messenger RNA (mRNA) and microRNA (miRNA) in sperm, which have important roles in regulating male fertility. Based on current trends in climate change, the incidence of chronically high temperatures that cause heat stress is expected to increase, posing increasing risks to health and survival of many species. The study of mRNAs and miRNAs can provide valuable insights to select animals that are more resilient to climate change. In addition to the search for more thermotolerant animals, other strategies to mitigate effects of heat stress include reproductive biotechniques and promotion of a better environment.

The neonatal increase in circulating luteinizing hormone (LH) is crucial for testicular development. In male pigs, blood LH levels start to increase approximately 1 week after birth and return to basal level by 5-6 weeks of age. This study tested the hypothesis that neonatal treatment with a combination of estrogens and androgens suppresses LH secretion and thereby inhibits testicular development. On Day 1 after birth, piglets received a slow-release implant containing estradiol (E, 8-40 mg) and trenbolone acetate (TBA, 40-200 mg) or remained intact. At 4 weeks of age, mean serum LH concentrations were ∼ 7 ng/mL in untreated males, whereas pigs with implants had serum LH concentrations < 1 ng/mL. Despite this reduction, LH was still detected in the pituitary glands of treated pigs. Interestingly, neonatal castration also lowered circulating LH, highlighting the importance of testis physiology in the early establishment of the reproductive axis. The higher dose (20 mg E2 + 100 mg TBA) inhibited testis function more effectively, as evidenced by lower circulating testosterone concentrations compared to intact pigs. Furthermore, E2 + TBA treatment had a lasting impact on testicular growth, resulting in smaller testes at 26 weeks of age and the presence of immature Leydig cells. Overall, neonatal E2 + TBA treatment suppressed the postnatal LH rise and testicular growth until market age, offering a potential non-surgical alternative to castration in male pigs.

Spermatogenesis is an extremely sophisticated and complex process and is regulated not only by a large number of genes, but also by a large number of epigenetic factors. Although existing studies have demonstrated that circRNAs plays an important regulatory role in spermatogenesis, there is still insufficient information to properly understand the regulatory role and mechanism of circRNA action. We addressed this issue by examining the testes of two Holstein bull developmental stages; three 8-week-olds (young bull, YB) and three 80-week-olds (adult bull, AB), randomly selected from the same breeding stock. A total of 3032 circRNAs, 683 miRNAs were identified as significantly differentially expressed noncoding RNAs, and 14,081 mRNAs. Based on these results, a circRNA-miRNA-mRNA competing endogenous RNA (ceRNA) regulatory network was constructed containing 3298 targeted regulatory axes. Modular analysis revealed a total of four modules in the ceRNA regulatory network. Functional analysis of these results showed that the ceRNA regulatory network in AB testis exhibited more positive regulatory effects on the spermatogenesis cycle checkpoints, chromosome and cytoplasm segregation, sperm tail formation, and sperm motility. In addition, screening combining the results of our previous studies on lncRNA regulation of spermatogenesis revealed 4 genes (FOXO4, PPP1CB, CDC26, and CDKN1B) that co-exist in the 2 ceRNA regulatory networks, lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA. A ceRNA regulatory network was constructed based on these genes. This study demonstrated the possible regulatory role of circRNAs in adult testicular spermatogenesis based on constructed transcriptome profiles and furtzher broadened our understanding of the regulatory role of circRNAs in spermatogenesis.